S137: SMALL EXTRACELLULAR VESICLES IN THE LEUKEMIA MICROENVIRONMENT SUSTAIN CLL PROGRESSION BY HAMPERING T CELL-MEDIATED ANTI-TUMOR IMMUNITY
Authors: E. Gargiulo, E. Viry, P. E. Morande, A. Largeot, S. Gonder, F. Xian, N. Ioannou, M. Benzarti, F. Kleine Borgmann, M. Mittelbronn, G. Dittmar, P. V. Nazarov, J. Meiser, B. Stamatopoulos, A. G. Ramsay, E. Moussay, J. Paggetti
Journal: HemaSphere (2022)

Background: Small extracellular vesicles (sEV) are nano-sized particles released by every cell and found in all biofluids. Given their composition and abundance, sEV are commonly involved in cell-to-cell communication through the transfer of genetic material and proteins. Furthermore, sEV possess direct functions carried out by sEV-ligands capable to affect the biological functions of targeted cells. In cancer, tumor-derived sEV are involved in the re-education of microenvironment (ME) cells promoting tumor proliferation, immune escape and metastasis. We previously demonstrated that leukemia-derived sEV are involved in the re-education of surrounding cells and increased immune escape. Indeed, chronic lymphocytic leukemia (CLL)-derived sEV induce stromal cell conversion into cancer-associated fibroblasts (Paggetti et al., Blood, 2015), and modulate PD-L1 expression in monocytes (Haderk et al. Science Immunology, 2017). Aims: The goal of the present work was to characterize leukemia ME-derived sEV (LME-sEV) and to evaluate their role in the disease development and progression in vivo. Methods: To obtain a biological representation of sEV in CLL microenvironment, we isolated LME-sEV directly from spleens of leukemic mice, obtaining a complex mix of sEV released by both CLL and ME cells alike. Small EV characterization was performed using a wide range of techniques, including qPCR, mass spectrometry and single sEV flow cytometry (FC). The effect on target cells was evaluated both ex vivo and in vivo using high-throughput techniques, FC, qPCR and cytotoxic assay. Small EV impact on CLL development in vivo was evaluated by generating a novel preclinical mouse model in which sEV release is genetically impaired due to Rab27a/b knock-out. Finally, we analyzed the expression of sEV-related genes in a cohort of 144 CLL patients using qPCR followed by regression analysis. Results: LME-sEV showed a distinct proteome (A) and RNA contents compared to healthy counterparts (HCME-sEV), including miRNA enriched in the plasma of CLL patients. Furthermore, FC-based immune checkpoint (ICP) screening showed the presence of multiple ICP ligands anchored on CLL-derived sEV (CD20+ subset of LME-sEV) (B), while high expression of the corresponding ICP receptors was found on T cells from matching LME. We also found that LME-sEV are internalized by different T cell subsets, thus we performed in vivo and ex vivo functional studies to assess sEV impact on T cells. High-throughput analysis of cells isolated from spleens of control mice treated with LME-sEV revealed considerable physiological changes mainly in CD8+ T cells. Indeed, CD8+ T cells showed alterations in their transcriptome, proteome and metabolome leading to cell exhaustion, decreased functions and survival. In line with this, absence of sEV dramatically delayed CLL progression in vivo. This effect was due to CLL inability to escape immune surveillance in absence of sEV and this was rescued by LME-sEV treatment (C). Finally, we identified a consistent sEV gene signatures in CLL patients correlating with treatment-free survival, overall survival, and with unfavorable clinical parameters routinely used in CLL diagnosis and prognosis (D). Image:Summary/Conclusion: By using different preclinical murine models and strategies, our results demonstrated for the first time that sEV in CLL ME play a key pro-tumoral role in leukemia development by negatively affecting the anti-tumor immune response. Furthermore, high expression of sEV-related genes correlated with poor survival and clinical parameters in CLL patients, suggesting sEV profiling as prognostic tool in CLL.